Erythrophagocytes in hemolytic anemia, wound healing, and cancer
Authors: Rok Humar, Dominik J. Schaer, Florence Vallelian
Journal: Trends in Molecular Medicine (2022)

Macrophages are the central hub for the detoxification of oxidative hemoglobin and heme. This protective function progresses either via engulfment and controlled degradation of red blood cells (RBCs) or via receptor-mediated clearance of RBC toxins.Intracellular heme signaling via BACH1, SPI-C, NRF2, and ATF1 orchestrates macrophage transformation into specialized erythrophagocytes with optimized antioxidant and iron recycling capacity.Novel discoveries demonstrated that erythrophagocytes have a unique anti-inflammatory phenotype with a disease- and context-dependent modulator effect on host defense, wound healing, atherosclerosis, and cancer progression.Enhancement or targeted suppression of erythrophagocyte functions may be exploited as novel therapeutic strategies. Hemolysis is a ubiquitous pathology defined as premature red blood cell destruction within the circulation or local tissues. One of the most archetypal functions of macrophages is phagocytosis of damaged or extravasated red blood cells, preventing the extracellular release of toxic hemoglobin and heme. Upon erythrophagocytosis, spiking intracellular heme concentrations drive macrophage transformation into erythrophagocytes, leveraging antioxidative and iron recycling capacities to defend against hemolytic stress. This unique phenotype transformation is coordinated by a regulatory network comprising the transcription factors BACH1, SPI-C, NRF2, and ATF1. Erythrophagocytes negatively regulate inflammation and immunity and may modulate disease-specific outcomes in hemolytic anemia, wound healing, atherosclerosis, and cancer. In this opinion article, we outline the known and presumed functions of erythrophagocytes and their implications for therapeutic innovation and research. Hemolysis is a ubiquitous pathology defined as premature red blood cell destruction within the circulation or local tissues. One of the most archetypal functions of macrophages is phagocytosis of damaged or extravasated red blood cells, preventing the extracellular release of toxic hemoglobin and heme. Upon erythrophagocytosis, spiking intracellular heme concentrations drive macrophage transformation into erythrophagocytes, leveraging antioxidative and iron recycling capacities to defend against hemolytic stress. This unique phenotype transformation is coordinated by a regulatory network comprising the transcription factors BACH1, SPI-C, NRF2, and ATF1. Erythrophagocytes negatively regulate inflammation and immunity and may modulate disease-specific outcomes in hemolytic anemia, wound healing, atherosclerosis, and cancer. In this opinion article, we outline the known and presumed functions of erythrophagocytes and their implications for therapeutic innovation and research. The term hemolysis describes the premature dysfunction and breakdown of red blood cells (RBCs) [1.Kaushansky K. et al.Williams Hematology. McGraw-Hill Education, 2016Google Scholar]. In blood, hemolysis results from either excessive clearance of altered RBCs by macrophages in the liver and spleen or rupture of membrane-dysfunctional RBCs within the circulation. Hemolytic anemias are caused by a broad range of genetic or acquired factors (see Clinician's corner). From a broader perspective, the pathophysiological concept of hemolysis and its downstream consequences should not be restricted to hemolytic anemias, as it also occurs ubiquitously secondary to RBC extravasation into tissues [2.Vallelian F. et al.Hemolysis, free hemoglobin toxicity and scavenger protein therapeutics.Blood. 2022; (Published online June 3, 2022)https://doi.org/10.1182/blood.2022015596Crossref PubMed Scopus (4) Google Scholar]. Examples of pathological RBC extravasation are numerous at the macroscopic and microscopic scales, including tissue trauma, spontaneous bleeding, such as subarachnoid hemorrhage, and in tissues containing immature and leaky blood vessels, such as tumors and large atherosclerotic plaques. In these conditions, RBC membranes become dysfunctional and closed tissue compartments are exposed to cell-free hemoglobin (Hb) and heme [2.Vallelian F. et al.Hemolysis, free hemoglobin toxicity and scavenger protein therapeutics.Blood. 2022; (Published online June 3, 2022)https://doi.org/10.1182/blood.2022015596Crossref PubMed Scopus (4) Google Scholar]. In the extracellular space, Hb and heme are toxins due to their uncontrolled biochemical reactivity with endogenous oxidants (Box 1 and Figure 1) [3.Schaer D.J. et al.Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins.Blood. 2013; 121: 1276-1284Crossref PubMed Scopus (462) Google Scholar,4.Gladwin M.T. et al.Hemolysis and cell-free hemoglobin drive an intrinsic mechanism for human disease.J. Clin. Invest. 2012; 122: 1205-1208Crossref PubMed Scopus (106) Google Scholar]. Physiological protection against cell-free Hb and heme toxicity is conferred by an evolutionary conserved and hierarchically organized system comprising macrophages and the scavenger proteins haptoglobin and hemopexin, which have a strong capacity to neutralize the adverse effects of cell-free Hb and heme, respectively [2.Vallelian F. et al.Hemolysis, free hemoglobin toxicity and scavenger protein therapeutics.Blood. 2022; (Published online June 3, 2022)https://doi.org/10.1182/blood.2022015596Crossref PubMed Scopus (4) Google Scholar,5.Buehler P.W. et al.Haptoglobin therapeutics and compartmentalization of cell-free hemoglobin toxicity.Trends Mol. Med. 2020; 26: 683-697Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar,6.Schaer D.J. et al.Haptoglobin, hemopexin, and related defense pathways-basic science, clinical perspectives, and drug development.Front. Physiol. 2014; 5: 415Crossref PubMed Scopus (183) Google Scholar].Box 1Systemic and localized hemolysis as a stressor of tissue homeostasisRed blood cells (RBCs) contain the oxygen carrier hemoglobin (Hb) and its non-amino acid component heme at concentrations exceeding 15 mM. The high concentration and the distinct intracellular environment of RBCs supports the unique structural and functional dynamics of the Hb tetramer, which can bind and release oxygen through a demand- and supply-regulated mechanism in the lung and metabolically active tissues. Hb and heme compartmentalization within RBCs protects the cardiovascular system and tissue microenvironments against its intrinsic toxicity. Hb and heme are highly reactive molecules. The biochemical reactivity of Hb and heme with the vasodilator nitric oxide, other endogenous oxidants, and unsaturated lipids defines their toxicity when present outside RBCs. The main adverse effects of cell-free Hb are vasoconstriction leading to cardiopulmonary dysfunction or ischemia, oxidation-driven tissue injury, and heme-ligation with Toll-like receptors (TLRs) to trigger inflammation [2.Vallelian F. et al.Hemolysis, free hemoglobin toxicity and scavenger protein therapeutics.Blood. 2022; (Published online June 3, 2022)https://doi.org/10.1182/blood.2022015596Crossref PubMed Scopus (4) Google Scholar] (see Figure 1 in main text). Additional toxic effects of heme-excess have been associated with intracellular heme-binding to cellular proteins comprising the proteasome [77.Vallelian F. et al.Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress.Cell Death Differ. 2015; 22: 597-611Crossref PubMed Scopus (37) Google Scholar] and cytoskeleton [78.Martins R. et al.Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions.Nat. Immunol. 2016; 17: 1361-1372Crossref PubMed Scopus (94) Google Scholar]. Red blood cells (RBCs) contain the oxygen carrier hemoglobin (Hb) and its non-amino acid component heme at concentrations exceeding 15 mM. The high concentration and the distinct intracellular environment of RBCs supports the unique structural and functional dynamics of the Hb tetramer, which can bind and release oxygen through a demand- and supply-regulated mechanism in the lung and metabolically active tissues. Hb and heme compartmentalization within RBCs protects the cardiovascular system and tissue microenvironments against its intrinsic toxicity. Hb and heme are highly reactive molecules. The biochemical reactivity of Hb and heme with the vasodilator nitric oxide, other endogenous oxidants, and unsaturated lipids defines their toxicity when present outside RBCs. The main adverse effects of cell-free Hb are vasoconstriction leading to cardiopulmonary dysfunction or ischemia, oxidation-driven tissue injury, and heme-ligation with Toll-like receptors (TLRs) to trigger inflammation [2.Vallelian F. et al.Hemolysis, free hemoglobin toxicity and scavenger protein therapeutics.Blood. 2022; (Published online June 3, 2022)https://doi.org/10.1182/blood.2022015596Crossref PubMed Scopus (4) Google Scholar] (see Figure 1 in main text). Additional toxic effects of heme-excess have been associated with intracellular heme-binding to cellular proteins comprising the proteasome [77.Vallelian F. et al.Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress.Cell Death Differ. 2015; 22: 597-611Crossref PubMed Scopus (37) Google Scholar] and cytoskeleton [78.Martins R. et al.Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions.Nat. Immunol. 2016; 17: 1361-1372Crossref PubMed Scopus (94) Google Scholar]. With their high capacity to clear damaged RBCs and cell-free Hb and heme, macrophages are the central hub for defense against the consequences of hemolytic toxin release. In the last few years, discoveries defined heme-driven signaling pathways endorsing a unique metabolic and immune-functional macrophage phenotype: erythrophagocytes (see Glossary). Erythrophagocyte transformation of macrophages enables adaptation to systemic or localized hemolytic stress. Their primary function is detoxifying heme in a controlled metabolic process, regulating local immunity, and translating environmental heme stimuli into a regenerative impetus. It is now emerging that depending on the disease context and the intensity, duration, and site of hemolysis, this unique function results in disease attenuation or, occasionally, disease acceleration. In this article, we summarize this research and outline a framework for the disease-modifying function of erythrophagocytes in hemolytic conditions. As the first line of defense against pathological hemolysis, macrophages recognize and engulf damaged but still intact RBCs and shuttle their toxic contents for controlled intracellular metabolic degradation [7.Theurl I. et al.On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver.Nat. Med. 2016; 22: 945-951Crossref PubMed Scopus (230) Google Scholar,8.Knutson M. Wessling-Resnick M. Iron metabolism in the reticuloendothelial system.Crit. Rev. Biochem. Mol. Biol. 2003; 38: 61-88Crossref PubMed Scopus (254) Google Scholar]. Macrophages also play essential roles in clearing cell-free Hb and complexes composed of Hb with haptoglobin and heme with hemopexin. Macrophages internalize these protein complexes specifically by receptor-mediated endocytosis via CD163 [9.Kristiansen M. et al.Identification of the haemoglobin scavenger receptor.Nature. 2001; 409: 198-201Crossref PubMed Scopus (1278) Google Scholar, 10.Schaer D.J. et al.CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin.Blood. 2006; 107: 373-380Crossref PubMed Scopus (226) Google Scholar, 11.Schaer C.A. et al.Constitutive endocytosis of CD163 mediates hemoglobin-heme uptake and determines the noninflammatory and protective transcriptional response of macrophages to hemoglobin.Circ. Res. 2006; 99: 943-950Crossref PubMed Scopus (212) Google Scholar] and CD91 [12.Hvidberg V. et al.Identification of the receptor scavenging hemopexin-heme complexes.Blood. 2005; 106: 2572-2579Crossref PubMed Scopus (331) Google Scholar] (Figure 1). 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In multiple independent observations across models of systemic hemolysis and more localized hemorrhages in wounds, atherosclerotic plaques, and cancer, researchers observed a conserved heme-induced macrophage phenotype with enhanced antioxidant and iron-recycling capacities as well as attenuated inflammatory and immune functions. These macrophages, which we later called erythrophagocytes, had a distinct gene expression profile that clearly deviated from the classical spectrum of IFN-gamma (M1) and IL-4 (M2) induced macrophage phenotypes in multiple ways [28.Pfefferlé M. et al.Hemolysis transforms liver macrophages into anti-inflammatory erythrophagocytes.J. Clin. Invest. 2020; 130: 5576-5590Crossref PubMed Scopus (20) Google Scholar]. Different observations reported progression of the heme-induced signal along various transcription factor networks, including BACH1, SPI-C, NRF2, and ATF1, ultimately converging in very similar phenotype characteristics. It is one of the unresolved questions in the field to determine the various mechanisms by which erythrophagocytosis and heme uptake activate these signaling cascades in a tissue–environmental context (see Outstanding questions). In the following sections, we will discuss how erythrophagocytes attenuate or exacerbate disease processes in a tissue- and disease type-specific context. To provide a systematic approach, we classify heme-polarized macrophages into central compartment erythrophagocytes in the liver and spleen and tissue-localized erythrophagocytes in atherosclerotic lesions, healing wounds, and in the tumor microenvironment. RBCs are cleared from the blood by central compartment macrophages in the spleen and liver. Under homeostatic conditions, the spleen is the main site of RBC turnover [7.Theurl I. et al.On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver.Nat. 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The RBC and its main constituent, Hb, are digested in phagolysosomes, then heme is exported into the cytosol of the erythrophagocyte by the heme transporter HRG1/SLC48A1 [16.White C. et al.HRG1 is essential for heme transport from the phagolysosome of macrophages during erythrophagocytosis.Cell Metab. 2013; 17: 261-270Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar,33.Rajagopal A. et al.Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins.Nature. 2008; 453: 1127-1131Crossref PubMed Scopus (221) Google Scholar]. Intracellular heme is a strong differentiation signal that drives RPM identity by binding and deactivating the transcriptional repressor BACH1, which controls heme response genes, including HMOX-1 and SPI-C [34.Sun J. et al.Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene.EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (512) Google Scholar,35.Ogawa K. et al.Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1.EMBO J. 2001; 20: 2835-2843Crossref PubMed Scopus (415) Google Scholar]. Activation of the transcription factor SPI-C by heme directs the differentiation of peripheral blood monocytes into RPMs; SPI-C-deleted mice are deficient in functional RPMs [36.Haldar M. et al.Heme-mediated SPI-C induction promotes monocyte differentiation into iron-recycling macrophages.Cell. 2014; 156: 1223-1234Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 37.Kohyama M. et al.Role for Spi-C in the development of red pulp macrophages and splenic iron homeostasis.Nature. 2009; 457: 318-321Crossref PubMed Scopus (304) Google Scholar, 38.Alam M.Z. et al.The heme connection: linking erythrocytes and macrophage biology.Front. Immunol. 2017; 8: 33Crossref PubMed Scopus (33) Google Scholar] (Figure 2, red panel). HMOX-1 is the archetypal erythrophagocyte enzyme that converts heme into iron, carbon monoxide, and biliverdin. Carbon monoxide can act as a signaling molecule and biliverdin is further reduced to bilirubin [17.Poss K.D. Tonegawa S. Heme oxygenase 1 is required for mammalian iron reutilization.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10919-10924Crossref PubMed Scopus (859) Google Scholar,18.Tenhunen R. et al.The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase.Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 748-755Crossref PubMed Scopus (1504) Google Scholar]. Depending on the homeostatic demand, iron is either stored intracellularly in a ferritin–iron complex or exported for binding to its plasma carrier protein transferrin, which shuttles iron to the bone marrow for erythropoietic recycling. Collectively, erythrophagocytes in the spleen support physiological RBC turnover and erythropoietic homeostasis (Figure 2, blue panel). When erythrolysis is enhanced over physiological RBC turnover in hemolytic anemia (see Clinician's corner), resident liver macrophages and recruited monocytes acquire erythrophagocytic function. Following this adaptive transformation, the pool of liver macrophages is the largest contributor to overall RBC clearance during pathological hemolysis, greatly exceeding the capacity of the spleen [7.Theurl I. et al.On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver.Nat. Med. 2016; 22: 945-951Crossref PubMed Scopus (230) Google Scholar]. On-demand erythrophagocyte recruitment into the liver occurs under the control of Csf-1 and is the most important mechanism for avoiding uncontrolled intravascular lysis of membrane-altered RBCs [7.Theurl I. et al.On-demand erythrocyte disposal and iron recycling requires transient macrophages in the liver.Nat. Med. 2016; 22: 945-951Crossref PubMed Scopus (230) Google Scholar,28.Pfefferlé M. et al.Hemolysis transforms liver macrophages into anti-inflammatory erythrophagocytes.J. Clin. Invest. 2020; 130: 5576-5590Crossref PubMed Scopus (20) Google Scholar]. Single-cell RNA sequencing analysis of liver macrophages from a genetic mouse model of spherocytosis defined the details of this macrophage phenotype [28.Pfefferlé M. et al.Hemolysis transforms liver macrophages into anti-inflammatory erythrophagocytes.J. Clin. Invest. 2020; 130: 5576-5590Crossref PubMed Scopus (20) Google Scholar]. In this model, erythrophagocytes display enhanced expression of antioxidant genes and genes involved in iron storage and export pathways. Moreover, and importantly, these macrophages show suppressed expression of major histocompatibility complex II (MHC class II), resistance against inflammatory activation by Toll-like receptor (TLR) agonists, interferon, and CD40 ligation, and impaired bacterial killing capacity [28.Pfefferlé M. et al.Hemolysis transforms liver macrophages into anti-inflammatory erythrophagocytes.J. Clin. 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Invest. 2020; 131e137468Google Scholar]. NRF2 is the principal activator of the oxidant stress response and was recently identified as a transcriptional repressor of inflammation-related genes [41.Taguchi K. et al.Molecular mechanisms of the Keap1–Nrf2 pathway in stress response and cancer evolution.Genes Cells. 2011; 16: 123-140Crossref PubMed Scopus (1059) Google Scholar,42.Kobayashi E.H. et al.Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription.Nat. Commun. 2016; 7: 11624Crossref PubMed Scopus (869) Google Scholar]. Therefore, a model comprising heme-signaling via NRF2 sufficiently explains the key phenotypic characteristics of erythrophagocytes (i.e., oxidative protection and suppressed inflammation and immune functions). Additional characteristics of erythrophagocytes are controlled by heme binding and inactivation of the transcriptional repressor BACH1, leading to strong activation of HMOX-1, and through post-transcriptional gene regulation by iron response elements (e.g., induction of ferritin and suppression of the transferrin receptor) [34.Sun J. et al.Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1 gene.EMBO J. 2002; 21: 5216-5224Crossref PubMed Scopus (512) Google Scholar,43.Rouault T.A. The role of iron regulatory proteins in mammalian iron homeostasis and disease.Nat. Chem. Biol. 2006; 2: 406-414Crossref PubMed Scopus (780) Google Scholar] (Figure 2, red panel). Besides the capacity to imprint a distinct macrophage phenotype in the liver, excessive heme exposure can also exert more profound effects on hematopoietic differentiation trajectories. Myeloid progenitors are the hematopoietic source of multiple immune cells, including monocyte-derived macrophages, granulocytes, and certain dendritic cells [44.Geissmann F. et al.Development of monocytes, macrophages, and dendritic cells.Science. 2010; 327: 656-661Crossref PubMed Scopus (2147) Google Scholar]. In cell culture differentiation experiments using bone marrow progenitors, the heme-enforced generation of on-demand erythrophagocytes occurred at the expense of the generation of myeloid-derived dendritic cells [45.Vallelian F. et al.Heme-stress activated NRF2 skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages in hemolytic anemia.Cell Death Differ. 2022; 29: 1450-1465Crossref PubMed Scopus (3) Google Scholar]. In mice with sickle cell disease or spherocytosis, this led to a hemolysis-induced dendritic cell deficiency in the spleen, which was accompanied by a reduced antigen presentation capacity and impaired CD4 T cell priming [45.Vallelian F. et al.Heme-stress activated NRF2 skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages in hemolytic anemia.Cell Death Differ. 2022; 29: 1450-1465Crossref PubMed Scopus (3) Google Scholar]. This adverse effect of systemic heme exposure may represent an important aspect of hyposplenism, which describes acquired immunodeficiency in patients with genetic hemolytic anemia, particularly sickle cell disease [46.Kato G.J. et al.Sickle cell disease.Nat. Rev. Dis. Primers. 2018; 4: 18010Crossref PubMed Scopus (256) Google Schol